Infliximab Therapeutic monitoring by tryptic peptide LC-MS/MS method improvements lead to improved accuracy with decreased imprecision and turnaround time

Introduction Therapeutic drug monitoring of infliximab has become the standard of care for inflammatory bowel disease in the setting of loss of response to therapy, and occasionally in proactive therapy personalization. Measurement of infliximab by tryptic peptide HPLC-MS/MS has been available since 2015, mostly in reference laboratories. Objectives Here, we present method improvements to our original published method leading to a more efficient, robust, and high throughput tryptic peptide HPLC-MS/MS assay for infliximab quantitation. Methods Deidentified residual serum samples submitted for clinical testing were used for method comparison and infliximab was spiked into normal human serum for performance studies. Improvements included the addition of a stable isotope labeled full length infliximab internal standard (IS) replacing a surrogate IS, and immunoenrichment using Melon Gel for immunoglobulins replacing the saturated ammonium sulfate precipitation. Digestion and chromatography were optimized, and automation was added. The method improvements were validated to include precision, accuracy, reportable range, linearity, and analytical sensitivity. Results The digestion time was reduced from overnight to 1 h. The assay analytical measuring range (AMR) remained the same throughout improvements, 1–100 µg/mL, with linearity of 0.98x + 0.50, R2 = 1.00. Intra- and inter-assay imprecision were less than 5 % CV at four different concentrations. Accuracy was assessed with 106 patients within the AMR; Passing-Bablok Regression yielded a slope of 1.00 and a y-intercept of 0.25. Turnaround time was reduced by 1 day, and imprecision of three levels of quality control trended down after new method implementation. Conclusions Method improvements including automation have allowed for assay completion in half a day, improving robustness and turnaround time.